1. Field of the Invention
The present invention relates to a sensor and a measuring chip assembly utilizing attenuated total reflection (ATR), such as a surface plasmon resonance sensor for quantitatively analyzing a substance in a liquid sample by utilizing surface plasmon excitation, and more particularly to a sensor and a measuring chip assembly, utilizing ATR, which employ a measuring chip equipped with one or a plurality of liquid-sample holding portions for holding a liquid sample.
2. Description of the Related Art
In metals, if free electrons are caused to vibrate in a group, a compression wave called a plasma wave will be generated. The compression wave, generated in the metal surface and quantized, is called a surface plasmon.
There have hitherto been proposed various kinds of surface plasmon resonance sensors for quantitatively analyzing a substance in a liquid sample by taking advantage of a phenomenon that the surface plasmon is excited by a light wave. Among such sensors, one employing a system called the “Kretschmann configuration” is particularly well known (e.g., see Japanese Unexamined Patent Publication No. 6(1994)-167443).
The surface plasmon resonance sensor employing the aforementioned system is constructed basically of a measuring chip, a light source for emitting a light beam, an optical system, and photodetection means. The measuring chip is equipped with a dielectric block; a thin film layer consisting of a metal film formed on the top surface of the dielectric block; and a liquid-sample holding portion for holding a liquid sample on the thin film layer. The dielectric block is formed, for example, into the shape of a prism. The optical system is used to make the light beam enter the dielectric block at various angles of incidence so that a condition for total internal reflection (TIR) is satisfied at the interface between the dielectric block and the thin film layer. The photodetection means measures the intensity of the light beam totally reflected at the interface, and detects the state of surface plasmon resonance, that is, the state of ATR.
To obtain various angles of incidence in the aforementioned manner, a relatively thin light beam is caused to strike the aforementioned interface at different angles of incidence, or a relatively thick light beam is caused to strike the interface convergently or divergently so that it includes components incident on the interface at various angles of incidence. In the former, the light beam whose reflection angle changes according to changes in the incidence angle thereof can be detected by a photodetector movable in synchronization with the reflection angle changes, or by an area sensor extending along the direction in which the reflection angle changes. In the latter, the light beams reflected at various angles can be detected by an area sensor extending in the direction where all the reflected light beams can be received.
In the surface plasmon resonance sensor mentioned above, if a light beam strikes the thin film layer at a specific incidence angle θsp greater than a critical incidence angle at which total internal reflection (TIR) takes place, an evanescent wave having electric field distribution is generated in a liquid sample in contact with the thin film layer. The evanescent wave excites surface plasmon at the interface between the thin film layer and the liquid sample. When the wave vector of the evanescent wave is equal to the wave number of the surface plasmon and therefore the wave numbers between the two are matched, the evanescent wave resonates with the surface plasmon and the light energy is transferred to the surface plasmon, whereby the intensity of the light totally reflected at the interface between the dielectric block and the thin film layer drops sharply. This sharp intensity drop is generally detected as a dark line by the aforementioned photodetection means.
Note that the aforementioned resonance occurs only when an incident light beam is a p-polarized light beam. Therefore, in order to make the resonance occur, there is a need to make settings in advance so that a light beam can strike the aforementioned interface as a p-polarized light beam.
If the wave number of the surface plasmon is found from the specific incidence angle θsp at which attenuated total reflection (ATR) takes place, the dielectric constant of a liquid sample to be analyzed can be calculated by the following Equation:Ksp(ω)=(ω/c){∈m(ω)∈s}½/{∈m(ω)+∈s}½where Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in vacuum, and ∈m and ∈s represent the dielectric constants of the thin film layer and the liquid sample, respectively.
If the dielectric constant ∈s of a liquid sample is found, the concentration of a specific substance in the liquid sample is found based on a predetermined calibration curve, etc. As a result, the dielectric constant of the liquid sample, that is, the properties of the liquid sample related to the refractive index thereof can be quantitatively analyzed by finding the specific incidence angle θsp at which the intensity of the reflected light at the interface drops sharply.
In addition, a leaky mode sensor is known as a similar sensor making use of ATR (for example, see “Spectral Researches,” Vol. 47, No. 1 (1998), pp. 21 to 23 and pp. 26 to 27). This leaky mode sensor is constructed basically of a measuring chip, a light source for emitting a light beam, an optical system, and photo detection means. The measuring chip is equipped with a dielectric block; a thin film layer consisting of a cladding layer formed on the top surface of the dielectric block and an optical waveguide layer formed on the cladding layer; and a liquid-sample holding portion for holding a liquid sample on the thin film layer. The dielectric block is formed, for example, into the shape of a prism. The optical system is used to make the light beam enter the dielectric block at various angles of incidence so that a condition for total internal reflection (TIR) is satisfied at the interface between the dielectric block and the cladding layer. The photodetection means measures the intensity of the light beam totally reflected at the interface, and detects the excited state of a waveguide mode, that is, the state of ATR.
In the leaky mode sensor mentioned above, if a light beam strikes the cladding layer through the dielectric block at incidence angles greater than a critical incidence angle at which total internal reflection (TIR) takes place, the light beam is transmitted through the cladding layer. Thereafter, in the optical waveguide layer formed on the cladding layer, only light with a specific wave number, incident at a specific incidence angle, propagates in a waveguide mode. If the waveguide mode is excited in this manner, most of the incident light is confined within the optical waveguide layer, and consequently, ATR occurs in which the intensity of light totally reflected at the aforementioned interface drops sharply. Since the wave number of the light propagating through the optical waveguide layer depends upon the refractive index of the liquid sample on the optical waveguide layer, the refractive index of the liquid sample and the properties of the liquid sample related to the refractive index can be analyzed by finding the aforementioned specific incidence angle θsp at which ATR takes place.
There are cases where in the field of pharmaceutical research, the above-mentioned surface plasmon resonance sensor and leaky mode measuring sensor are employed in the research of the interaction between a desired sensing substance and a liquid sample. For instance, the sensors are employed in the measurement of interaction, such as the bonding reaction between a specific substance contained in a liquid sample and a sensing substance and the dissociating reaction of a specific substance into a liquid sample from a bonded substance. Such interaction includes protein-protein interaction, DNA-protein interaction, sugar-protein interaction, protein-peptide interaction, lipid-protein interaction, a bond between chemical substances, and so on.
In addition, there are cases where the above-mentioned surface plasmon resonance sensor and leaky mode measuring sensor are used in a random screening method for detecting a specific substance that bonds to a sensing substance. In this case, a sensing substance is fixed on the aforementioned thin film layer. Then, a liquid sample containing various target substances is dropped into the sensing substance, and each time a predetermined time elapses, the state of ATR is measured.
If a target substance in the liquid sample bonds to the sensing substance, the refractive index of the sensing substance changes with the lapse of time by the bond therebetween. Therefore, the state of ATR is measured at predetermined intervals and it is measured whether a change has occurred in the state of ATR. In this manner, it can be decided whether there is a bond between a target substance and a sensing substance, that is, whether the target substance is a specific substance that bonds to the sensing substance. As a combination of a specific substance and a sensing substance, there are a combination of an antigen and an antibody and a combination of an antibody and an antibody. For example, a rabbit antihuman immunoglobulin G (IgG) antibody is fixed to a measuring chip as a sensing substance, and a human IgG antibody is employed as a specific substance.
Note that the present applicant has proposed a sensor, utilizing ATR, for making measurements by employing a measuring chip which has a plurality of liquid-sample holding portions (see Japanese Patent Application No. 2001-397411). The use of such a sensor makes it possible to measure a plurality of samples in a short time.
However, in the conventional sensors utilizing ATR which have been proposed, the state of ATR described above is measured after a liquid sample is supplied to a cup-shaped or dish-shaped liquid-sample holding portion having a thin film layer previously formed on the inner bottom surface thereof.
Because of this, when a measurement is made after the supply of a liquid sample, there is a possibility that (1) the liquid sample will evaporate, (2) the concentration of the liquid sample will change, and (3) the accuracy of the measurement of ATR will be reduced.
Particularly, in the case of measuring a temporal change in the interaction between the sensing substance and liquid sample in a measuring chip, measurements are often made for extensive periods of time and therefore there is a possibility that during the measurements, the liquid sample will evaporate and an accurate measurement of the temporal change cannot be made.